Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Cloverleaf representations of a human tRNA Gln UUG with all uridines (U) replaced by Ψ (blue) or m 1 Ψ (violet). Melting temperatures (°C) of tRNA Gln UUG with various modifications (Tm is indicated). Biological replicates, N = 3. ( B ) 2D representation of the structure of tRNA with site-specific Ψ (circles) added by individual PUS. PUS are shown in cartoon representation: PUS1, PUS3, and PUS4 are obtained from Alphafold2 prediction while PUS7 (PDB 5KKP) and PUS10 (PDB 2V9K) are crystal structures. ( C ) Detection of PUS-dependent Ψ formation on tRNA Gln UUG . The reverse‐transcribed cDNA products were resolved in a 15% urea gel and the CMC‐Ψ mediated short cDNAs product are indicated by double-arrows (Ψ 13 orange, Ψ 28 blue, Ψ 39 green, Ψ 55 red) while the Cy5-primer is indicated by a triangle. A short exposure of the same gel is shown in the dashed box. The Ψ 13 and Ψ 28 -corresponding cDNAs are detected using a different set of site-specific primers (shown in a dash lined box on the right). ( D ) Melting temperature changes (ΔTm, °C) of tRNA Gln UUG , tRNA Glu UUC , tRNA Asp GUC by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( E ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( F ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC modified only by PUS4, only by PUS7, by PUS4 and PUS7 and after mixing the individual PUS4 and PUS7 samples at a 1:1 ratio (left). Melting temperature changes (ΔTm, °C) of tRNA Gly CCC after mixing samples modified individually by PUS4 or PUS4/7 at different ratios (middle). Melting temperature changes (ΔTm, °C) of tRNA Gly CCC after mixing samples modified individually by PUS7 or PUS4/7 at different ratios (middle). Biological replicates, N = 3. ( G ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC , after incubation with PUS4 (left) PUS7 (middle) and PUS4/7 (right) for the indicated time in comparison to corresponding tRNA not incubated with any PUS. Detection of PUS-dependent Ψ formation on tRNA Gly CCC (below). Biological replicates, N = 3. ( H ) Melting temperature changes (ΔTm, °C) of unmodified (left) and PUS3, PUS4, and PUS7 modified (right) tRNA Gly CCC , after repeated heating and cooling cycles. Biological replicates, N = 3. The distribution of the data is represented in box plots. The central line inside the box indicates the median and the square the mean. The lower and upper edges correspond to the first (Q1) and third (Q3) quartiles, respectively, defining the interquartile range (IQR). The whiskers extend to the minimal and maximal values within the 1.5 IQR. Points beyond this range are considered outliers and are shown as transparent points. All statistical analyses were performed using one-way ANOVA ( α = 0.05) with a Bonferroni multiple comparisons test. Statistically significant differences are indicated (ns: no significance, **** p ≤ 0.0001). Inactive PUS variants are indicated by *. .

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Reverse Transcription, Comparison, Modification, Incubation

Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Cartoon presentation of domains of each PUS (upper-left). The length is labeled, and domain is highlighted where the catalytic residue is indicated by a triangle. The truncated regions of PUS1 and PUS7 are shown as dashed lines. An SDS-PAGE gel of recombinant Hs PUS enzymes (lower-left). Catalytic mutants marked with stars. SEC profiles of purified human PUS enzymes (upper-right) and the collected fraction for each purification were resolved in SDS-PAGE gels (lower-right). ( B ) MST analyses of PUS, including wild type and inactive form (DA), binding to tRNA Gln UUG . The calculated Kds of wild-type PUS and inactive forms (DA), binding to tRNA Gln UUG . ( C , D ) Detection of PUS-dependent Ψ formation on tRNA Gln UUG , tRNA Gly CCC , tRNA Glu UUC and tRNA Asp GUC . The reverse‐transcribed cDNA products were resolved in a 15% or 18% urea gel and the CMC‐Ψ mediated short cDNAs are indicated by double-arrows (Ψ 13 orange, Ψ 27/28 blue, Ψ 39 green, Ψ 54 yellow Ψ 55 red). Each tRNA primer (labeled with Cy5) is indicated by a triangle. In the case of tRNA Asp GUC , the signal for primer is obtained from a short exposure shown in the dash lined box while the Ψ 13 -dependent cDNA is obtained using a site-specific primer (shown in a dash lined box on the side). ( E ) Cryo-EM reconstructions of unmodified and Ψ 13 - and Ψ 39 -modified tRNA Gln UUG . The modified sites are highlighted by color code in the 2D cartoon and the model (Ψ 13 orange and Ψ 39 green).

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Labeling, Residue, SDS Page, Recombinant, Purification, Binding Assay, Reverse Transcription, Cryo-EM Sample Prep, Modification

Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Melting temperature changes (ΔTm, °C) of tRNA His GUG , tRNA Lys UUU , tRNA Ser UGA , tRNA Arg UCU by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( B ) Local resolution estimations for tRNA Gln UUG . The relative resolution scale is shown in the inset. Ψ sites are highlighted by circles (Ψ 13 orange, Ψ 39 green, Ψ 55 red). The arrows indicate the local conformational changes. All cryo-EM maps are contoured to RMSD = 8. ( C ) tRNA representation with Ψ sites and the positions of Cy5 probe are highlighted (green). Global melting temperatures (Tm, °C) measured with RiboGreen for unlabeled and U 48 or 5ʹ-Cy5 labeled tRNA Gln UUG . Melting temperature changes (ΔTm, °C) of tRNA Gln UUG labeled with Cy5 at 5ʹ (left) or U 48 (right) upon Ψ modifications. The temperature (Tm) scale is shown in the inset. Biological replicates, N = 3. Inactive PUS are indicated by *. The distribution of the data is represented in box plots. The central line inside the box indicates the median and the square the mean. The lower and upper edges correspond to the first (Q1) and third (Q3) quartiles, respectively, defining the interquartile range (IQR). The whiskers extend to the minimal and maximal values within the 1.5 IQR. Points beyond this range are considered outliers and are shown as transparent points. All statistical analysis was performed using one-way ANOVA (α = 0.05) with a Bonferroni multiple comparisons test. Statistically significant differences are indicated (ns: no significance, **** p ≤ 0.0001. For tRNA Ser UGA * p = 0.01517, for tRNA Arg UCU * p = 0.04746). .

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Comparison, Cryo-EM Sample Prep, Labeling